The present invention relates to the fields of stem cells and gene therapy.
Cell proliferation in the adult mammalian brain is ubiquitous but is largely confined to the measured production of glia. Except for discrete regions in the hippocampus and the subventricular zone (SVZ), neurogenesis is conspicuously absent. The reasons why these areas continue to generate neurons are unknown, but primary cell cultures from the adult rodent brain are beginning to provide some insights. Cultures initiated from adult SVZ or hippocampal tissues contain proliferative neuronal and glial-restricted progenitors as well as multipotent precursors with the characteristics of neural stem cells, i.e., the ability to self-renew and the ability to generate both neurons and glia.
It has been suggested that stem cells may be more widely distributed since cells from non-neurogenic areas repeatedly passaged in the presence of high concentrations of basic fibroblast growth factor (FGF-2) appear to begin to generate neurons in vitro. This observation is consistent with the isolation of neuronal progenitors from these areas, but the protracted times in culture suggests another explanation. It is known that stem cell cultures initiated from hippocampal tissues will spontaneously transform, due to accumulated genetic abnormalities. Abnormalities in chromosome number can occur in as little as 30 population doublings and, as cells become increasingly aneuploid, it is possible that glial-restricted progenitors acquire capabilities beyond those available in vivo.
With existing methodologies, it has been difficult to distinguish between the activation of a latent potential vs. in vitro mutation. Unlike fibroblast tissues, which are easily dissociated and yield relatively abundant precursor populations, adult tissues yield few progenitors and the progenitor preparations are contaminated with differentiated cells and tissue debris. The myelin-rich debris inhibits cell attachment and growth while differentiated cells complicate the evaluation of lineage potential in acutely isolated cultures. Past studies have evaluated xe2x80x9cprogenitorsxe2x80x9d only after repeated passaging had eliminated the debris and differentiated cells. Even if these cells had remained diploid, they may have been dramatically altered in prolonged culture. Accordingly, there exists a need in the art for isolating a xe2x80x9ccleanxe2x80x9d (i.e., more enriched) cell population containing putative progenitor and stem cells, so that they can be studied without contaminating debris.
The ability to isolate progenitor or stem cells from a variety of tissues would provide a basis for therapeutic applications using these immature cell types. Neural tissue, in particular, comprises a unique biological system that presents unique therapeutic challenges. Damaged neural tissue has proven very difficult to repair or replace. To facilitate the repair or replacement of neural tissue, scientists have focused their efforts on the identification, isolation and use of neuronal stem cells (or xe2x80x9cprogenitor cellsxe2x80x9d). With an appropriately pluripotent neural progenitor or stem cell, regeneration or augmentation of a variety of neural cell types is a possibility. Indeed, with a progenitor cell exhibiting an even more widely ranging plasticity, the regeneration or augmentation of a variety of cell types should be possible. Similarly, the stem cell would be a useful vehicle for introducing exogenous genetic material, as desired to achieve therapeutic results.
Occular tissue, in particular the retina, represents a highly specialized neural structure for which repair is often required. For example, the eye is frequently subjected to environmentally or genetically induced injury. As a result, appropriately plastic stem cells would present a valuable vehicle for repair, replacement and/or genetic manipulation (e.g., gene therapy). Although physical damage to the eye may require merely replacing damaged cells with a cell type exhibiting the required plasticity, genetically mediated degeneration of occular tissue presents a more complex challenge.
In many instances, the exact molecular mechanisms that mediate occular or retinal degeneration are poorly understood. Grafting genetically modified cells provides an effective method for evaluating the relative impact of candidate molecules on retinal biology. In addition, the ability to introduce engineered cells may provide a considerable therapeutic benefit for a variety of progressive degenerative diseases. Past attempts to use cell grafts for the delivery of transgene products in the eye have met with mixed success. Heterotypic grafts of non-neural cells fail to integrate and often physically disrupt the normal retinal architecture; Planck, S. R., et al. Curr. Eye Res. 11: 1031 (1992). Homotypic fibroblast tissue grafts show some integration but are not amenable to genetic manipulation prior to implantation. See, for example, Seiler, M. J. and Aramant, R. B., Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina, Exp. Brain Res. 105: 59-66 (1995); Gouras, P., Du, J., Kjeldbye, H., Yamamoto, S., Zack, D. J., Long-term photoreceptor transplants in dystrophic and normal mouse retina, 35:3145-3153 (1994); and Gouras, P., Du, J., Kjeldbye, H., Kwun R., Lopez, R., Zack, D. J., Transplanted photoreceptors identified in dystrophic mouse retina by a transgenic reporter gene, Invest Ophthalmol. Vis. Sci. 32: 3167-3174 (1991). Grafts of retinal cell lines (i.e., immortalized cell lines) partially overcome these problems. Trisler, D., Rutin, J. and Pessac, B., Retinal engineering: engrafted neural cell lines locate in appropriate layers, Proc. Natl. Acad. Sci. U.S.A. 93: 6269-274 (1996); del Cerro, M., Notter, M. F., Seigel, G., Lazar, E., Chader, G., del Cerro, C., Intraretinal xenografts of differentiated human retinoblastoma cells integrate with the host retina, Brain Res., 583:12-22 (1992). However, immortalized cells present potential risks of tumor formation that makes their use less than ideal.
Several groups have reported heterotypic transplants of neuronal progenitor cells, immortalized neural cell lines or embryonic neural precursors into the CNS. To date, however, none of these studies has shown satisfactory diversity and distribution of cell types to render such cells broadly useful either as cell source for cell replacement therapy or-for the expression of transgenes in the host tissue.
In recent years, significant attention has been directed toward identification and characterization of very immature progenitor cells in the adult brain. Gage, F. H., Ray, J. and Fisher, L. J., Isolation, characterization, and use of stem cells from the CNS, Annu. Rev. Neurosci., 18:159-192 (1995). Additionally, it has been reported that stem cell-like multipotent progenitors can be isolated from adult hippocampus of rats, expanded in vitro and subsequently grafted into adult hippocampus and olfactory bulb where they demonstrate site-specific neuronal differentiation. See, for example, Palmer, T. D., Talkahashi, J., Gage, F. H., The rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci., 8: 389 (1997); Palmer, T. D., Ray, J. and Gage, F. H. FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain, Mol. Cell Neurosci. 6: 474-486 (1995); Gage, F. H., Coates, P. W., Palmer, T. D., et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. U. S. A. 92:11879-11883 (1995); and Suhonen, J. O., Peterson, D. A., Ray,J. and Gage, F. H. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo, Nature 383:624-627 (1996). Some of the phenotypes generated in the olfactory bulb are not found in the hippocampus, suggesting that the most immature of these stem-like cells may retain considerable plasticity.
As part of the central nervous system, both developmentally and phenotypically, the retina shares the recalcitrance of brain and spinal cord with respect to functional repair. This is unfortunate in that, among heritable conditions alone, there are over 100 examples of diseases involving the loss of retinal neurons. Bird, A. C., Retinal photoreceptor dystrophies L. I. Edward Jackson Memorial Lecture, see comments. Am J Ophthalmol 119, 543-62, (1995); Simunovic, M. P., and Moore, A. T., The cone dystrophies. Eye 12,.553-65 (1998). One strategy for replacing these cells has been to transplant retinal tissue from healthy donors to the retina of the diseased host (see, for example, Gouras, P., Du, J. , Kjeldbye, H., Yamamoto, S., and Zack, D. J., Long-term photoreceptor transplants in dystrophic and normal mouse retina. Invest Ophthalmol Vis Sci 35, 3145-53 (1994); Silverman, M. S., and Hughes, S. E., Transplantation of photoreceptors to light-damaged retina. Invest Ophthalmot Vis Sci 30, 1684-90 (1989)). While the results of such studies have been encouraging in terms of graft survival, the problem of integration between graft and host has proved to be daunting. The graft-host interface is often well demarcated histologically, with ultrastructural studies revealing the presence of a dense glial scar across which few neurites are seen to cross. Ivert, L., Gouras, P., Naeser, P., and Narfstrom, K., Photoreceptor allografts in a feline model of retinal degeneration. Graefes Arch Clin Exp Ophffidmol 236, 844-52 (1998).
Thus, there exists a need to develop methods for the isolation and exploitation of stem cells which can, in turn, be used as a therapeutic agent and/or as a vehicle for additional manipulation of gene-based therapeutics in order to treat damaged or diseased tissue, particularly neuronal tissue, and more particularly ocular tissue.
The present invention is directed to methods of enriching a cell population for stem cells and/or stein cell precursors. Stem cells isolated by the present invention are pluripotent, however, precursor stem cells are not. In another embodiment of the present invention there is provided a method for treating precursor stem cells in a manner that produces stem cells. Stem cells, whether isolated by invention methods or generated by invention methods are useful in repairing damaged or diseased, specialized or differentiated tissue in mature animals, particularly neuronal tissue such as retinas.
One embodiment of the present invention relates to transplantation of adult, hippocampus (HC)-derived progenitor cells into a selected neural tissue site of a recipient. These cells can functionally integrate into mature and immature neural tissue. The invention encompasses, in one aspect, repopulating a retina of a dystrophic animal with neurons, by injecting clonally derived, adult central nervous system derived stem cells (ACSC) derived from a healthy donor animal into an eye of the dystrophic recipient. Herein disclosed is the first successful and stable integration of clonally derived ACSC/ACPC into same-species but different strain recipients (e.g., Fischer rat-derived adult hippocampal derived progenitor cells (AHPCs) into dystrophic RCS rats). Surprisingly, AHPCs were also found to integrate successfully into a xenogeneic recipient (e.g., rat AHPCs into the retina of dystrophic rd-I mice).
In accordance with the present invention there is provided a method for obtaining Adult Mammalian Stem Cells or Adult Mammalian Progenitor stem Cells from tissue (AMSC and AMPC, respectively), said method comprising subjecting dissociated mammalian tissue to one or more buoyancy-based separation systems. In one aspect of this embodiment, there is provided a method for obtaining adult mammalian CNS-derived progenitor cells (ACPC) or Adult Mammalian CNS-derived Stem Cells (ACSC) from a cell population containing adult mammalian central nervous system (CNS) tissue, said method comprising subjecting dissociated mammalian CNS tissue to one or more buoyancy-based separation systems.
In another embodiment of the present invention there are provided adult mammalian stem cells or adult mammalian progenitor cells isolated by invention methods. In a specific aspect of this embodiment, there are provided adult CNS-derived progenitor cells (ACPC) or adult mammalian CNS-derived stem cells (ACSC) isolated by subjecting dissociated mammalian CNS tissue to one or more buoyancy-based separation systems.
In another embodiment of the present invention AMSC and AMPC have the ability to adapt to a heterotypic environment.
As used herein, xe2x80x9cstem cellsxe2x80x9d means cells that are self-renewing and multipotent (i.e., that are not lineage restricted). Stem cells includes AMSC and ACSC as defined herein. Stem cells are characterized as both self-renewing and able to differentiate. xe2x80x9cProgenitorxe2x80x9d or xe2x80x9cprecursorxe2x80x9d cells means an undifferentiated cell whose lineal descendants differentiate along the appropriate pathway to produce a fully differentiated phenotype (i.e., cells with a restricted lineage). For example, neural stem cells isolated from the hippocampus (HC) or the subventricular zone, are self renewing and able to generate, in vitro, multiple types of cells including neurons, glia and even hematopoetic cells. In contrast, neural progenitor or precursor cells are lineage restricted and while self-renewing, only generate glia in vitro. Progenitor or precursor cells include AMPC and ACPC, as described herein.
As used herein, xe2x80x9csubventricular zonexe2x80x9d or xe2x80x9csubventricular residuumxe2x80x9d means a thin lamina extending inward about 50 xcexcm from the ependymal surface, including the hippocampus alveus but excluding ependymal cells.
AMSC can be characterized as self-renewing and able to generate (i.e., differentiate into) mature, differentiated cells of the tissue type from which the cells were isolated, and the like, either in vivo, or in vitro when grown in mitogen free media. Similarly, Adult Mammalian CNS-derived Stem Cells (ACSC) can be characterized as self-renewing and able to generate (i.e., differentiate into) neurons, glia, hematopoetic cells, and the like, either in vivo, or in vitro when grown in mitogen free media.
Adult Mammalian derived Progenitor Cells (AMPC) can be characterized as self-renewing and able to generate (i.e., via replication) mature, differentiated cells of the tissue type from which the cells were isolated, and the like, either in vivo, or in vitro when grown in mitogen supplemented media. Similarly, Adult Mammalian CNS-derived Progenitor Cells (ACPC) can be characterized as self-renewing and able to generate neurons either in vivo, or in vitro when grown in mitogen supplemented media. Moreover, these ACPC are further characterized as being able to generate neurons, glia and hematopoetic cells in vitro when grown in the presence of mitogen, e.g., FGF, or the like.
Accordingly, in another embodiment of the present invention, there are provided methods for obtaining AMSC by growing AMPC in the presence of FGF-2. In one aspect of this embodiment, there is provided a method for obtaining ACSC by growing ACPC in the presence of a FGF, preferably FGF-2, 4, 6, or 8; more preferably FGF-2 or 4 and most preferably FGF-2.
For discussion purposes, as used hereinafter, xe2x80x9cadult mammalian derived stem cells (AMSC)xe2x80x9d includes ACSC and xe2x80x9cadult mammalian derived progenitor cells AMPCxe2x80x9d includes ACPC.
Invention AMSC and AMPC can be derived from any tissue, including CNS, heart, liver lung, bone marrow, and the like.
CNS tissue from which invention ACPC can be derived include whole brain, hippocampus, spinal cord, cortex, striatum, cerebellum, thalamus, hypothalamus, amigdyla, basal forebrain, ventral mesencephalon, optic nerve, locus cerleus, and the like. Indeed, it is expected that any tissue can yield progenitor and stem cells if processed in the manner described herein. In a presently preferred embodiment, invention ACSC/ACPC are isolated from the hippocampus, more preferably from the adult hippocampus.
As used herein with respect to stem cells of the present invention, xe2x80x9cheterotypic environmentsxe2x80x9d to which the cells are able to adapt include all non-source, or non-native, neural tissue such as whole brain, hippocampus, spinal cord, cortex, striatum, cerebellum, thalamus, hypothalamus, amigdyla, basal forebrain, ventral mesencephalon, optic nerve, locus ceruleus, and the like, as well as CNS associated tissues such as eye tissues, the vitreous of the eye, and the like. In addition, heterotypic environments include in vitro culture systems in which the foregoing cell types and lineages derived therefrom are cultured.
In one embodiment of the present invention, the xe2x80x9cability to adaptxe2x80x9d comprises the ability of invention AMSC to respond to temporal and/or spatial cues of the heterotypic environment, either in vivo, in vitro, or both. These temporal and spatial cues include very broad classes of compounds known to have regulatory effects on cells, including those that provide differentiation signals, and the like. More particularly, as used herein, xe2x80x9ctemporal cuesxe2x80x9d refers to compounds and conditions provided by the heterotypic environment in a time-dependent manner, including development stage associated compounds, cell cycle associated compounds and conditions, as well as combinations of any two or more thereof.
As used herein, xe2x80x9cspatial cuesxe2x80x9d include compounds and conditions provided by the heterotypic environment in a location specific manner, including any molecule or compound found in the heterotypic environment that provides cell-differentiation signals, such as trophic factors, hormones, cognate receptors for the foregoing, and the like, as well as combinations thereof.
As used herein, the term xe2x80x9ctrophic factorxe2x80x9d refers to compounds with trophic actions that promote and/or control proliferation, differentiation, migration, survival and/or death (e.g., apoptosis) of their target cells. Such factors include cytokines, neurotrophins, growth factors, mitogens, co-factors, and the like, including epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, schwanoma-derived growth factor, glial growth factor, stiatal-derived neuronotrophic factor, platelet-derived growth factor, hepatocyte growth factor, scatter factor (HGF-SF), transforming growth factor-beta and related molecules, neurotransmitters, and hormones. Those of ordinary skill in the art will recognize additional trophic factors that can be employed in the present invention (see, e.g., Aebischer et al. Neurotrophic Factors (Handbook of Experimental Pharmacology, Vol 134) (Springer Verlag, 1998); Meyers, R. A. Encyclopedia of Molecular Biology and Molecular Medicine: Denaturation of DNAxe2x80x94Growth Factors (VCH Pub, 1996); Meager and Robinson, Growth Factors: Essential Data (John Wiley and Sons, 1999); McKay and Brown, Growth Factors and Receptors: A Practical Approach (Oxford University Press, 1998); Leroith and Bondy, Growth Factors and Cytokines in Health and Disease, Vol 1A and 1B: A Multi-Volume Treatise (JAI Pr, 1996); Lenfant et al., Growth Factors of the Vascular and Nervous Systems: Functional Characterization and Biotechnology: International Symposium on Biotechnology of Grow (S. Karger Publishing, 1992).
xe2x80x9cTrophic factorsxe2x80x9d have a broad range of biological activities and their activity and specificity may be achieved by cooperation with other factors. Although trophic factors are generally active at extremely low concentrations, high concentrations of mitogen together with high cell density are often required to induce proliferation of multipotent neural progenitor cell populations. For example, growth factors for early progenitors may be useful for enhancing the viability of progenitor cells as well as treating disorders by renewal of mature cells from the progenitor cell pool.
Preferred trophic factors contemplated for use in the present invention are mitogenic growth factors, like fibroblast growth factor-2 (FGF-2) (Gage, F. H., et al., 1995, Proc. Natl Acad. Sci. USA 92:11879-11883) and epidermal growth factor (EGF) (Lois, C., and Alvarez-Buylla, A., 1993, Proc. Natl. Acad. Sci. USA 90(5):2074-2077), which induce proliferation and/or propogation of progenitor cells, e.g., neural progenitor cells isolated from the brain. Studies from single cells in culture demonstrate that FGF-2 (Gritti, A., et al., 1996, J. Neurosci. 16:1091-1100) and EGF (Reynolds, B. A., and Weiss, S., 1996, Develop. Biol. 175:1-13) are mitogens for multipotent neural stem cells and likely cooperate with other trophic factors (Cattaneo, E., and McKay, R., 1990, Nature 347:762-765; Stemple, D. L., and Anderson, D. J., 1992, Cell 71:973-985), some of which are yet unknown (Davis, A. A., and Temple, S., 1994, Nature 372:263-266; Temple, S., 1989, Nature 340:471-473; Kilpatrick, T. J., and Bartlett, P. F., 1993, Neuron 10:255-265; Palmer, T. D., et al., 1997, Mol. Cell. Neurosci. 8:389-404) to achieve specificity.
Hormones that provide spatial cues include thyroid hormone and the like. Receptors include the steroid/thyroid hormone superfamily of receptors, neurotrophin receptors TrkB and TrkC, and the like.
The temporal and spatial cues described herein may be provided to invention cells as either molecules that are supplied exogenously (i.e., extracellularly) or endogenously (e.g., through the expression of native and/or introduced nucleic acids encoding such molecules, and the like).
Invention AMSC are self-renewing (i.e., are capable of replication to generate additional AMSC). In addition, invention AMSC/AMPC, due to their pluripotent character, are capable of exhibiting a wide variety of responses upon exposure to a heterotypic environment with its associated temporal and spatial bio-information (i.e., cues). Because invention AMSC/AMPC are pluripotent, in one embodiment of the present invention AMSC/AMPC response to a heterotypic environment includes differentiation into a more lineage restricted type of cell found in the tissue from which the AMSC/AMPC was isolated. Accordingly, because invention ACSC/ACPC are also pluripotent stem cells, in one embodiment of the present invention, ACSC/ACPC response to a heterotypic environment includes differentiation into neurons, and glia, including astroglia and/or oligodendroglia, and the like.
As a result of the remarkable ability of invention AMSC/AMPC to adapt to a variety of heterotypic environments with the concomitant ability to integrate and differentiate, they are excellent candidates for gene therapy applications. Accordingly, in another embodiment of the present invention, there are provided AMSC/AMPC containing one or more heterologous DNA sequences (e.g., transgenes, and the like). In a presently preferred embodiment, the AMSC/AMPC are capable of expressing proteins encoded by the heterologous DNA sequences.
As described herein, invention AMSC/AMPC are able to integrate and differentiate into a number of different tissue types. Invention ACSC/ACPC are able to integrate and differentiate primarily into neural tissues. As such, invention AMSC/AMPC are useful as therapeutic agents for replacing or augmenting diseased or damaged tissue. Invention AMSC/AMPC may, however, also carry and express heterologous DNA sequences. Thus, in accordance with the present invention there are provided methods of therapy comprising administering to a patient in need thereof a cell population comprising modified AMSC/AMPC, such as, for example, those described herein, in an amount sufficient to provide a desired therapeutic effect. As those of skill in the art will understand, an amount sufficient to provide a therapeutic effect will vary according to the condition being treated, the locus of introduction, the level of enrichment for AMSC/AMPC in the donor cell population, the presence in donor AMSC/AMPC of transgenes, the relative level of expression of any such transgene(s), and the like. Accordingly, the individual practitioner may be required to take such factors into account when proceeding with a therapeutic regimen in accordance with the present invention. In one embodiment of the present invention, a therapeutically effective amount is an amount effective for introducing or complementing one or more missing and/or defective genes, wherein the gene(s) so introduced comprise heterologous genetic material contained and expresed within said AMSC/AMPC and their descendants.
Although the retina originates from the neural tube, the optic vesicle forms early in development and the retina becomes regionally isolated and highly specialized. Given this spatial and temporal separation, it would seem unlikely that ACSC/ACPC could be used to replace retinal neurons, yet these immature cells retain sufficient adaptability to integrate within the normal retina and provide a means to deliver gene products to the eye. Thus, by placing adult hippocampal stem cell and progenitors (i.e., ACSC/ACPC isolated from the adult hippocampus) into the developing and adult eye in accordance with the present invention, these cells were found to be surprisingly well suited for gene delivery. When grafted to the developing retina, adult hippocampal stem cell-progenitors (AHPCs) were broadly integrated within most layers of the retina and acquired striking morphological similarities to Mxc3xcller, astroglia, bipolar, amacrine, horizontal and photoreceptor cells. Not only do these results demonstrate the considerable adaptability of adult-derived neural stem cells but also demonstrate the first successful attempt to nondestructively insert engineered normal-diploid cells into the complex architecture of the optic retina.
ACSC/ACPC are capable of reaching all layers of the retina, and differentiating into cells with local phenotypic characteristics. These cells represent an exciting new tool for studying and manipulating retinal development in mammalian species. Given that they can be propagated in vitro and, following transplantation, can extensively repopulate an actively degenerating retina in visually mature animals, this invention is also useful in treating retinal diseases involving neuronal cell loss. In view of the results discussed herein, it is expected that ACSC/ACPC will similarly be able to differentiate into the appropriate neuronal cell lineage of other neural sites into which these progenitors are transplanted in vivo. Therefore, ACSC/ACPC transplantation is also useful to treat other neurological diseases and injuries involving neuronal loss or damage. Similarly, AMSC/AMPC can be used to treat diseases and injuries to the type of tissue from which the cells were isolated.
Thus, in another embodiment of the present invention there are provided methods for the transplantation of ACPC into dystrophic neural tissue. In application, the invention encompasses a method of treating dystrophic neural tissue, comprising introducing ACPC derived from an adult animal donor into dystrophic neural tissue in an animal recipient, e.g., by grafting or applying adult progenitor cells into tissue affected by the disorder.
The recipient may be an young (immature) animal or an adult (mature) animal. The ACPC donor and recipient may be of different species (xenogeneic). Exemplary donor-recipient pairs include, but are not limited, to: a donor rat and a recipient mouse; a donor mouse and a recipient rat; a donor pig and a recipient human. The donor and recipient may be of the same species (e.g., human-to-human, rat-to-rat, mouse-to-mouse), and be allogeneic (of different strains, i.e., have different histocompatibility genes) or syngeneic (of the same strain, i.e., having identical histocompatibility genes).
Examples of dystrophic neural tissue that can be treated by the invention include the central nervous system (CNS) and neural tissue of the eye, particularly the retina or optic nerve. Thus, in another embodiment, the invention encompasses a method of repopulating or rescuing a dystrophic retina with neural cells, comprising introducing neural progenitor cells derived from an adult donor (e.g., ACPC or ACSC) into dystrophic neural tissue of an animal recipient. The method is particularly useful for treating dystrophic retinal tissue caused by an optic neuropathy, e.g., glaucoma.
As used herein, the term xe2x80x9cdystrophic neural tissuexe2x80x9d encompasses damaged, injured, or diseased neural tissue, which neutral tissue includes differentiated neural tissue. Thus the present invention provides methods for treating a neuronal or neural disorder or neural injury. A xe2x80x9cneuronal disorderxe2x80x9d or xe2x80x9cneural disorderxe2x80x9d is any disorder or disease that involves the nervous system. One type of neuronal disorder is a neurodegenerative disorder. Neurodegenerative disorders include but are not limited to: (1) diseases of central motor systems including degenerative conditions affecting the basal ganglia (e.g., Huntington""s disease, Wilson""s disease, Striatonigral degeneration, corticobasal ganglionic degeneration, Tourettes syndrome, Parkinson""s disease, progressive supranuclear palsy, progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration, cerebral angiopathy (both hereditary and sporadic)); (2) diseases affecting sensory neurons (e.g., Friedreich""s ataxia, diabetes, peripheral neuropathy, retinal neuronal degeneration); (3) diseases of limbic and cortical systems (e.g., s cerebral amyloidosis, Pick""s atrophy, Retts syndrome; (4) neurodegenerative pathologies involving multiple neuronal systems and/or brainstem (e.g., Alzheimer""s disease, AIDS-related dementia, Leigh""s disease, diffuse Lewy body disease, epilepsy, Multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, late-degenerative stages of Down""s syndrome, Alper""s disease, vertigo as result of CNS degeneration; (5) pathologies arising with aging and chronic alcohol or drug abuse (e.g., with alcoholism the degeneration of neurons in locus oeruleus, cerebellum, cholinergic basal forebrain; with aging degeneration of cerebellar neurons and conical neurons leading to cognitive and motor impairments; and with chronic amphetamine abuse degeneration of basal ganglia neurons leading to motor impairments; and (6) pathological changes resulting from focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy, hyperglycemia, hypoglycemia or direct trauma.
The presence of a neuronal or neurodegenerative disorder or injury may be indicated by subjective symptoms, such as pain, change in sensation including decreased sensation, muscle weakness, coordination problems, imbalance, neurasthenia, malaise, decreased reaction times, tremors, confusion, poor memory, uncontrollable movement, lack of affect, obsessive/compulsive behavior, aphasia, agnosia, visual neglect, etc. Frequently, objective indicia, or signs observable by a physician or a health care provider, overlap with subjective indicia. Examples of objective indicia include the physician""s observation of signs such as decreased reaction time, muscle fasciculations, tremors, rigidity, spasticity, muscle weakness, poor coordination, disorientation, dysphasia, dysarthria, and imbalance. Additionally, objective signs can include laboratory parameters, such as the assessment of neural tissue loss and function by Positron Emission Tomography (PET) or functional Magnetic Resonance Imaging MRI), blood tests, biopsies and electrical studies such as electromyographic data.
xe2x80x9cTreatingxe2x80x9d dystrophic neural tissue is intended to encompass repairing, replacing, augmenting, rescuing, or repopulating the diseased or damaged neural tissue, or otherwise compensating for the dystrophic condition of the neural tissue.
xe2x80x9cIntroductionxe2x80x9d of ACPC/ACMC or ACSC/ACPC into dystrophic neural tissue (e.g., a damaged or diseased retina or optic nerve), may be accomplished by any means known in the medical arts, including but not limited to grafting and injection. It should be understood that such means of introducing the neural progenitor cells also encompass placing, injecting or grafting them into a site separate and/or apart from the diseased or damaged neural tissue site, since the neural progenitor cells are capable of migrating to and integrating into that dystrophic site. For example, dystrophic retinal or optic nerve tissue can be treated by placing neural progenitor cells into the vitreous of the eye.
In another aspect of the foregoing embodiment of the present invention, there are provided therapeutic methods comprising administering to a patient in need thereof an amount of ACSC/ACPC effective to repair or replace defective, damaged or dead tissue. In a presently preferred embodiment, cells which are to be added to or replaced comprise optic cells, including, retinal cells, Mxc3xcller cells, amacrine cells, bipolar cells, horizontal cells, photoreceptors, astroglial cells, and the like.
Because of the pluripotent nature of invention AMSC/AMPC, and the resulting multiplicity of loci where such cells may be introduced in order to achieve therapeutic effects, there is a broad range of tissue damage and disease states which can be treated using invention AMSC/AMPC. Many disease states (e.g., liver disease) result in damaged or necrotic tissue. These types of diseases are ideal for replacement or augmentation therapy comprising the administration of invention AMSC/AMPC. The plastic and pluripotent nature of AMSC/AMPC make them ideal candidates for their use as a source of cells which can be used to replace or correct for cells lost in disease or injury, even in the absence of exogenous genetic material. For example, invention AMSC/AMPC can be used to replace a variety of tissue types throughout the body that are encompassed within the different phenotypes that progeny of AMSC/AMPC can exhibit, upon differentiation, including glial cells, neurons, and the like. Accordingly, in a particular embodiment of the present invention, there are provided therapeutic methods comprising administering to a patient in need thereof a cell population comprising AMSC/AMPC as described herein, in an amount sufficient to provide a therapeutic effect. In one aspect of the present invention, expression of AMSC/AMPC native genes in the cell population occurs as necessary for AMSC/AMPC to proliferate and differentiate in order to replace or add cells of a desired type.
The therapeutic benefit of the invention can be evaluated or assessed by any of a number of subjective or objective factors indicating a response of the condition being treated. Such indices include measures of increased neural or neuronal proliferation or more normal function of surviving brain areas. In addition, macroscopic methods of evaluating the effects of the invention can be used which may be invasive or noninvasive. Further examples of evidence of a therapeutic benefit include clinical evaluations of cognitive functions including object identification, increased performance speed of defined tasks as compared to pretreatment performance speeds, and nerve conduction velocity studies.
In another aspect of the invention, the neural progenitor cells have preferably been cultured in vitro in a culture medium comprising at least one trophic factor, or even combinations of such factors.
As used herein, the neural progenitor cells can be cultivated in the presence of a trophic factor, or combinations of trophic factors. For example, these cells can be cultivated in medium having xe2x80x9cneurotrophinsxe2x80x9d (or xe2x80x9cneurotrophic factorxe2x80x9d) that promote the survival and functional activity of nerve or glial cells, including a factor that enhances neural differentiation, induces neural proliferation, influences synaptic functions, and/or promotes the survival of neurons that are normally destined to die, during different phases of the development of the central and peripheral nervous system. Exemplary neurotrophins include, for example, ciliary neurotrophic factor (CNF), nerve growth factor (NGF), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), glia derived neurotrophic factor (GDNF), and the like. Such factors are characterized by their trophic actions, their expression patterns in the brain, and molecular aspects of their receptors and intracellular signaling pathways. Neurotrophic factors that have been identified include NT4, NT-5, NT-6, NT-7, ciliary neuronotrophic factor (CNTF), Glial cell line-derived neurotrophic factor (GDNF), and Purpurin. Neuron-specific enolase (NSE) has been found to be a neuronal survival factor. Other factors possessing a broader spectrum of functions, which have neurotrophic activities but are not normally classified as neurotrophins, also are contemplated for use in the invention. These xe2x80x9cneurotrophin-like factorsxe2x80x9d include epithelial growth factor (EGF), heparin-binding neurite-promoting factor (HBNF), IGF-2, a-FGF and b-FGF, PDGF, neuron-specific enolase (NSE), and Activin A. Other factors have been identified which specifically influence neuronal differentiation and influence transmitter phenotypes without affecting neuronal survival. Although the intracerebral administration of FGF-2 has been shown to stimulate neurogenesis in the adult rat SVZ, FGF-2 alone in the adult rat hippocampus has a limited effect on the proliferation of neural stem/progenitor cells (Kuhn et al. (1997); Wagner et al. (1999) each herein incorporated by reference).
In a preferred embodiment of the present, the present invention employs FGF and FGF-like factors, including a-FGF, b-FGF such as FGF-2, FGF4, FGF6, and the like. A particularly advantageous medium for culturing neural progenitor cells comprises one of the following: fibroblast growth factor (FGF) alone (particularly basic FGF or FGF-2), FGF plus epidermal growth factor (EGF), or FGF plus EGF plus heparin, which is mitogenic.
In view of the foregoing observations, and in view of the observation that many tissues have latent poppulations of progenitor and/or stem cells, in another embodiment of the present invention, there are provided methods for inducing the proliferation and differentiation of stem and progenitor cells in situ. Thus, in one aspect of this embodiment, a therapeutically effective amount of agent comprising one or more of a neurotrophic factor (i.e., nuerotrophin), a mitogen, a neuotrophin-like factor, or the like is administered to a subject with damaged, diseased or dystrophic tissue (e.g., neuronal tissue such as retina, or the like). It is presently preferred that the agent be directly administered to the affected tissue via injection, topical application, or the like.
Some disease states are characterized by one or more defective or missing genes. Such diseases are ideally treated by the adminstration of invention AMSC/AMPC containing one or more transgenes. Thus, in another embodiment of the present invention, there are provided therapeutic methods as described herein, wherein one or more disease associated transgenes incorporated and expressed in said invention AMSC/AMPC. Examples of neuronal tissue-associated disease states and their associated genes include Huntingtons Corea (one or more of gamma amino butyric acid (GABA) decarboxyalse and ciliary neurotrophic factor (CNTF)), Alzheimer""s disease (one or more of acetylcholinesterase, neuronal growth factor (NGF), brain derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF)), Parkinson""s disease (one or more of tyrosine hydroxylase, DOPA decarboxylase, DMAT, GDNF, BDNF and FGF), amyotropic lateral sclerosis (CNTF), and the like.
While invention AMSC/AMPC are useful to introduce therapeutic genes, it may be desirable to introduce into a host or patient one or more genes that are not strictly therapeutic but which may be useful in other ways, for example, as tracking genes (i.e., markers), as genes to induce migration, as genes to induce mitosis, as survival genes, as suicide genes, and the like. Marker genes contemplated for use in the practice of the present invention include genes encoding a modified green fluorescent protein (GFP) derived from jellyfish, xcex2-Galatosidase (the LacZ gene product), neomycin phosphotransferase (neo), Luciferase, and the like.
As will be recognized by those of skill in the art, a variety of methods exist for the introduction of genetic material into cells such as invention AMSC/AMPC. Such methods include viral and non-viral methods. Non-viral methods contemplated for use in the practice of the present invention include electroporation, microinjection, polyethylene glycol precipitation, high velocity ballistic penetration by small particles with the nucleic acid to be introduced contained either within the matrix of such particles, or on the surface thereof (Klein, el al., Nature 327:70, 1987), or the like. Viral methods contemplated for use in the practice of the present invention include the use of retroviral vectors, and the like. It is presently preferred that retroviral vectors be employed for introducing genetic material into invention AMSC/AMPC. In one aspect of the present invention, replication deficient vectors are employed. Such vectors are well known to those of skill in the art.
Numerous examples of disease exist that are suitable for treatment with invention AMSC/AMPC. Some of these disease states are equally suited for treatment using invention AMSC/AMPC with and/or without incorporated transgenes. For example, the liver plays a central role in the pathophysiology of many inherited metabolic diseases. Although the adult liver has the unusual ability to regenerate after injury, the liver is an important target for cell therapy. Therefor, in another embodiment of the present invention AMSC/AMPC are introduced into the liver where they differentiate into hepatocytes, and replace dead and dying cells, thereby correcting disease phenotypes. When particular diseases are associated with one or more missing or defective genes, such diseases are treatable with invention AMSC/AMPC wherein the missing/defective gene(s) is/are incorporated.
Recent experimental data from immune and endocrine studies using spontaneous or transgenic models of diabetes have emphasized the role of islets of Langerhans, and particularly beta cells, in autoimmune insulin-dependent (Type 1) diabetes mellitus (IDDM) pathogenesis. IDDM is a chronic disorder that results from the destruction of the insulin-producing beta cells of the pancreatic islets. Accordingly, in another aspect of the present invention, AMSC/AMPC can be grafted in the pancreas for the replacement of damaged pancreas cells with the grafted cells. When particular diabetic pathologies are associated with one or more missing or defective genes, such pathologies are treatable with invention AMSC/AMPC wherein the missing/defective gene(s) is/are incorporated.
Duchenne muscular dystrophy (DMD) is characterized by slow and progressive muscle weakness affecting limb and respiratory muscles, which degenerate until fatal cardiorespiratory failure. Myodystrophy of the Duchenne type results from mutations affecting the gene for dystrophin, a cytoskeletal protein. A form of congenital dystrophy caused by a deficiency of the a2 subunit of the basement membrane protein laminin/merosin is termed Merosin-Deficient Congenital Muscular Dystrophy (MCMD). Accordingly, in another aspect of the present invention, AMSC/AMPC are grafted into muscles wherein they differentiate to become myoblasts and replace degenerating muscle cells.
Cardiac disease, typified in many instances by damaged heart muscle, is another target for cell replacement. Accordingly, in yet another aspect of the present invention, AMSC/AMPC are transplanted into the heart to replace diseased cells and improve heart function.
Pulmonary disease (i.e., Cystic fibrosis) is the most common autosomally inherited disease, and is caused by the defective gene Cftr, which encodes an ion channel at the cell membrane. Augmentation of lung tissue with AMSC/AMPC can alleviate the reduced respiratory function caused by the defective genotype. Accordingly, in still another aspect of the present invention, AMSC/AMPC are grafted into the lung in order to replace the diseased cells having defective ion channels, and restore normal lung function. As a heritable disorder, this disease is also an ideal candidate for treatment using invention ACSC/ACPC with appropriately incorporated Cftr-augmenting exogenous nucleic acids.
As readily understood by those of skill in the art, the most direct method for administration of invention AMSC/AMPC to the desired site is likely to be by injection, however, any means of administering cells that results in correct localization and integration is contemplated for use in the practice of the present invention.
As those of skill in the art will understand, a number of factors may be determinative of when and how a stem or progenitor cell differentiates. As a result, it may be desirable to induce differentiation of invention AMSC/AMPC in a controlled manner and/or by employing factors which are not easily or desirably introduced into the locus of therapeutic AMSC/AMPC introduction. Accordingly, in another embodiment of the present invention, there are provided therapeutic methods as described herein, wherein said AMSC/AMPC have been induced to differentiate, prior to administration to the subject, by in vitro exposure to extracellular and/or intracellular factors described herein, including trophic factors, cytokines, mitogens, hormones, cognate receptors for the foregoing, and the like, as well as combinations of any two or more thereof.
As will be appreciated by those of skill in the art, proper isolation and treatment of source tissues for AMSC/AMPC is desirable in order to obtain a population of cells comprising AMSC/AMPC. ACSC/ACPC are used as a paradigm for the present disclosure, however, any tissue type may be employed to isolate corresponding AMSC/AMPC. Thus, while a whole brain or other source neuronal tissue, as described herein, all comprise ACSC/ACPC, it is desirable for therapeutic purposes to provide a cell population containing primarily ACSC/ACPC and lacking a substantial amount of other cell types and/or debris. Accordingly, it is presently preferred that cell populations be enriched for ACSC/ACPC. This enrichment can be carried out by a number of methods. Thus, in accordance with one embodiment of the present invention there are provided methods for enriching a cell population containing adult mammalian CNS tissue for adult mammalian CNS-derived stem cells (ACSC), said method comprising subjecting dissociated mammalian CNS tissue to one or more separation systems. Although it is contemplated that, with proper execution, any separation system can be adapted to isolate ACSC/ACPC from CNS tissue, separation systems contemplated for use in the practice of the present invention include buoyancy-based separation systems, charge-based separation systems, fluorescent activated cell sorting systems (FACS), and the like, as well as combinations thereof.
It is presently preferred that bouyancy based separation systems be employed in the practice of the present invention. In one embodiment of the present invention, the bouyancy based separation system employed is density gradient centrifugation. Density gradients can be crated using any suitable media, including, PERCOLL(trademark) (polyvinylpyrrolidone-coated silica colloids), FICOLL(trademark) (copolymers of sucrose and epichlorohydrin), sucrose, and the like. In an even more preferred embodiment of the present invention, a PERCOLL(trademark) (polyvinylpyrrolidone-coated silica colloids) gradient is employed. Invention AMSC/AMPC will typically have a density in the range of about 1.06 up to about 1.08 g/ml; and more typically a density in the range of about 1.072 up to about 1.075 g/ml. Thus, in a presently preferred embodiment of the present invention, the PERCOLL(trademark) (polyvinylpyrrolidone-coated silica colloids) gradient employed in the practice of the present invention is an approximately 50% PERCOLL(trademark) (polyvinylpyrrolidone-coated silica colloids) gradient. As will be understood by those of skill in the art, the gradient can be modified to take into account the bouyant density of the particular stem cells being sought (e.g., hepatic stem cells, or the like).
The invention will now be described in greater detail by referring to the following non-limiting examples.